RNA editing plays a critical role in the expression of certain gene products by generating proteins not encoded in the gene sequence. One type of RNA editing involves the conversion of adenosine residues into inosine. This A-to-I RNA editing is carried out by multiple members of an emerging gene family, ADAR (adenosine deaminases acting on RNA). Three separate ADAR gene family members (ADAR1-3) which display significant differences in their substrate and editing site selectivity have been identified in humans and rodents. Analysis of staged ADAR1 null mutant and chimeric mouse embryos revealed that most embryos died at midgestation stage with defects in the erythropoiesis system. The results indicate that abnormal proliferation and/or differentiation of erythroid cells is caused by underediting of the RNA of currently unknown ADAR1 target gene(s). In this application, we propose to determine the molecular basis of erythrogenic defects observed with ADAR1 null mutant embryos. First, we will conduct a series of experiments to distinguish whether abnormal erythropoiesis is caused by cell-autonomous defects, or abnormalities in the fetal liver microenvironment. Mice or embryos harboring a new ADAR1 null mutation allele (ADAR1del) and an embryonic liver-specific ADAR1 null mutation allele (ADAR1flox/Alb:AFP-Cre) established recently by the Cre- loxP recombination system will be used for in vitro assay of erythroid progenitor cell differentiation and in utero transplantation experiments. ADAR1 del/del (-/-) homozygous ES cell lines will be used for in vitro differentiation experiments and also for chimeric mice formation and tissue contribution analysis. ADAR1 null erythroid cells generated by in vitro culture of gene targeted ES cells and/or ADAR1 null fetal livers will then be used for identification and cloning of ADAR1 target genes (new A-to-I RNA editing sites) critical for embryonic erythroid maturation. The information gained from the proposed experiments will allow us to better understand the physiological significance of A-to-I RNA editing and the role played by ADAR1 in the regulation of erythropoiesis during development. Our research may reveal critical information leading to new strategies for therapeutic intervention for certain human dyserythropoietic disorders.